Synthesis of coumarin derivatives and investigation of their inhibitory effects on lung cancer cell motility

Based on the pharmaceutical potentials of coumarins, which have antitumor activity, we synthesized new coumarin derivatives and evaluated their biological activities. The new coumarin derivatives were chemically synthesized from 4-hydroxycoumarin, and their structures were confirmed by nuclear magnetic resonance data. Ten of the synthesized compounds were investigated for antimetastatic activity against lung carcinoma cells. Several of the tested compounds showed good to mild inhibitory effects on lung cancer cell motility. There were no cytotoxic effects related to the use of these compounds. 4-Hydroxycoumarin derivatives, 4h and 4i, elicited the significant inhibitory effect on lung cancer cell motility by suppressing expression of the epithelial–mesenchymal transition markers N-cadherin, Snail, and Twist.


Scientific Reports
| (2022) 12:21635 | https://doi.org/10.1038/s41598-022-26212-z www.nature.com/scientificreports/ Biological evaluation. Cancer metastasis accounts for much of the morbidity and is associated with ~ 90% of the cancer-associated deaths 19 . According to the GLOBOCAN analysis, lung cancer is one of the most malignant types with metastasis, with a higher mortality rate of around 18.4% 20 . The motility of cancer cells contributes to tumor metastasis with several stages, including invading across the basement membranes, migration to blood, intravasation and movement into distant organs 21 . Cell migration and invasion are relevant phenotypes when studying the effect of novel therapeutic drugs against metastasis in cancer development and progression 22 . This study aimed to elucidate whether chemically synthesized coumarin derivatives affect lung cancer cell motility. To determine the cytotoxic effects of all the compounds on lung cancer cells, viability of A549, H460, and H1975 cells were evaluated via the MTT assays. Treatment with 3.125-20 μM of 4-hydroxycoumarin and its derivatives 4a-4j did not affect the viabilities of A549, H460, and H1975 cells. Treatment with 50-100 μM of 4-hydroxycoumarin and its derivatives 4a-4j inhibited the cell viabilities by ~ 25% in A549, H460, and H1975 cells ( Fig. 3a and Supplementary Fig. S1). An invasion assay was performed to examine their effects on A549 cell motility. Cells were treated with 4-hydroxycoumarin and its derivatives 4a-4j at nontoxic concentration of 5 μM for 24 h. Treatment with compounds 4d, 4h, and 4i significantly inhibited A549 cell invasion by ~ 40%, ~ 50%, and ~ 40% (p < 0.0001), respectively (Fig. 3b,c). To elucidate how these three compounds inhibit cancer cell motility at noncytotoxic concentrations, cells were mainly treated with nontoxic concentrations at 5, 10, or 15 μM of compounds 4d, 4h, and 4i in subsequent experiments. Treatment with compounds 4d, 4h, and 4i dose-dependently decreased the number of invaded A549 cells (Fig. 4).
Wound healing assays are a commonly used method to evaluate cell migration ability 23 . The wound healing assay was performed by treating A549 cells with 15 μM compounds 4d, 4h, and 4i (Fig. 5). Cell migration was inhibited by treatment with compounds 4h and 4i in a time-dependent manner but was unaffected by compounds 4d treatment.
Moreover, the migration assay was performed using the other lung cancer cell lines H460, and H1650. Treatment with compounds 4h and 4i significantly decreased abilities of migrated cells in both cell lines (Fig. 6a,b). However, cell migration abilities of H460 and H1650 were also unaffected by treatment with compound 4d (Supplementary Fig. S2), indicating that it might not be a selection against lung cancer motility. The invasion assays were performed using H460, H1650, and H1975 cells. Treatment with compounds 4h and 4i significantly decreased numbers of invaded cells of all three cell lines by more than 25% (p < 0.005) (Fig. 6c). Taken together, these results indicate that compounds 4h and 4i significantly suppress lung cancer cell motility.
The EMT plays a role in lung cancer cell motility and metastasis 24 . In the EMT process, epithelial cells lose cell-cell adhesion and decrease the expression of epithelial cells marker such as E-cadherin, inversely, the expression of mesenchymal cell markers such as N-cadherin is increased, associated with invasive phenotype 25 . Several reports have proved that loss of E-cadherin expression or induction of N-cadherin led to poor prognosis in lung cancer [26][27][28] . E-cadherin and N-cadherin, as well as their regulators Snail and Twist, are indicators of the EMT 24 . The protein and mRNA expression levels of these EMT markers and their regulators were examined to determine how compounds 4h and 4i suppress lung cancer cell motility. Treatment with 15 μM compounds 4h decreased expression of not only N-cadherin but also E-cadherin and suppressed mRNA expression of Snail and Twist (Fig. 7a,c). Treatment with compound 4i significantly increased the expression of E-cadherin and downregulated the expression of N-cadherin, Snail, and Twist at a dose-dependent manner (Fig. 7b,d). These results indicate   www.nature.com/scientificreports/ that compound 4i inhibits expression of EMT markers more than compound 4h. However, compound 4d did not affect E/N-cadherin expression as shown in Supplementary Fig. S2c. Taken together, our data demonstrate that compounds 4h and 4i inhibit lung cancer cell motility by downregulating EMT markers.

Conclusion
Ten amidocoumarin derivatives (4a-4j) were newly synthesized by a chemical reaction, and their inhibitory effects on lung carcinoma were evaluated. Compounds 4h and 4i inhibited invasion and migration of lung cancer cells by modulating expression of EMT effectors. Moreover, the inhibition of EMT markers by compound 4i was highest, suggesting that it is a potential inhibitor of lung cancer motility. A further biological study is required to elucidate the detailed underlying molecular mechanism.
Statistical analysis. All in vitro experiments were performed in triplicate. Data are presented as the mean standard deviation (SD) of 3 independent experiments with 3 replicates each. The student's t-test was utilized to determine statistical significance between two groups, and analysis of variance was utilized between three or more groups, respectively. Values of p < 0.05 were considered statistically significant. All statistical analyses were performed using Sigma Plot 12.5 software (Systat software, Erkrath, Germany).

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information file.